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Prehypertension is highly prevalent worldwide.1 It is esti-mated that ≈30% to 50% of the population have this con-

dition. It frequently complicates other cardiometabolic risk factors and is closely associated with coronary heart disease, stroke, and renal dysfunction.2 Early intervention in prehyper-tension substantially prevents the incidence of hypertension and related damage to target organs. Currently, several strategies are used to treat prehypertension, including the incorporation of therapeutic lifestyle changes, such as healthy dietary intake and regular physical activity, as well as the use of antihyperten-sive drugs, such as an angiotensin II receptor blocker. Although these treatments improve prehypertension, poor compliance and limitations associated with antihypertensive medications prevent their application in the general population. Thus, there

is an urgent need to identify reliable and accurate measures to prevent the development of prehypertension.

Taurine (2-aminoethanesulfonic acid) is the most abun-dant, semiessential, sulfur-containing amino acid. It can be synthesized in vivo by cysteine in the presence of cysteine dioxygenase,3 but taurine is mainly acquired from dietary sources, such as eggs, meat, and seafood. Hydrogen sulfide (H

2S) is synthesized from 2 sulfur-containing amino acids,

l-cysteine and l-methionine, by the 3 enzymes, cystathionine-γ-lyase (CSE), cystathionine-β-synthetase (CBS), and 3-mer-captopyruvate sulfurtransferase.4

Taurine has several potentially beneficial cardiovascular effects that involve regulation of the nitric oxide system and endothelial function,5,6 the renin–angiotensin–aldosterone

Abstract—Taurine, the most abundant, semiessential, sulfur-containing amino acid, is well known to lower blood pressure (BP) in hypertensive animal models. However, no rigorous clinical trial has validated whether this beneficial effect of taurine occurs in human hypertension or prehypertension, a key stage in the development of hypertension. In this randomized, double-blind, placebo-controlled study, we assessed the effects of taurine intervention on BP and vascular function in prehypertension. We randomly assigned 120 eligible prehypertensive individuals to receive either taurine supplementation (1.6 g per day) or a placebo for 12 weeks. Taurine supplementation significantly decreased the clinic and 24-hour ambulatory BPs, especially in those with high-normal BP. Mean clinic systolic BP reduction for taurine/placebo was 7.2/2.6 mm Hg, and diastolic BP was 4.7/1.3 mm Hg. Mean ambulatory systolic BP reduction for taurine/placebo was 3.8/0.3 mm Hg, and diastolic BP was 3.5/0.6 mm Hg. In addition, taurine supplementation significantly improved endothelium-dependent and endothelium-independent vasodilation and increased plasma H

2S and taurine concentrations.

Furthermore, changes in BP were negatively correlated with both the plasma H2S and taurine levels in taurine-treated

prehypertensive individuals. To further elucidate the hypotensive mechanism, experimental studies were performed both in vivo and in vitro. The results showed that taurine treatment upregulated the expression of hydrogen sulfide–synthesizing enzymes and reduced agonist-induced vascular reactivity through the inhibition of transient receptor potential channel subtype 3–mediated calcium influx in human and mouse mesenteric arteries. In conclusion, the antihypertensive effect of chronic taurine supplementation shows promise in the treatment of prehypertension through improvement of vascular function. (Hypertension. 2016;67:541-549. DOI: 10.1161/HYPERTENSIONAHA.115.06624.) • Online Data Supplement

Key Words: blood pressure ■ hydrogen sulfide ■ prehypertension ■ taurine ■ transient receptor potential channels

Received October 9, 2015; first decision October 22, 2015; revision accepted December 14, 2015.From the Department of Hypertension and Endocrinology, Center for Hypertension and Metabolic Diseases, Daping Hospital, Third Military Medical

University, Chongqing Institute of Hypertension, Chongqing, China.*These authors contributed equally to this work.The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.

115.06624/-/DC1.Reprint requests to Zhiming Zhu or Daoyan Liu, Department of Hypertension and Endocrinology, Center for Hypertension and Metabolic Diseases,

Daping Hospital, Third Military Medical University, Chongqing Institute of Hypertension, Changjiang Zhilu, no. 10, Chongqing 400042, China. E-mail [email protected] or [email protected]

Taurine Supplementation Lowers Blood Pressure and Improves Vascular Function in Prehypertension

Randomized, Double-Blind, Placebo-Controlled Study

Qianqian Sun,* Bin Wang,* Yingsha Li,* Fang Sun, Peng Li, Weijie Xia, Xunmei Zhou, Qiang Li, Xiaojing Wang, Jing Chen, Xiangru Zeng, Zhigang Zhao, Hongbo He, Daoyan Liu,

Zhiming Zhu

© 2016 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.115.06624

Clinical Trial

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542 Hypertension March 2016

system,7,8 the oxidative stress system and sympathoadrenal activity,9 and the endoplasmic reticulum stress system.10,11 Epidemiological studies have demonstrated a reduction in plasma sulfur amino acids in hypertensive patients.12 Several clinical studies have reported that diets rich in taurine can reduce cardiovascular risks regardless of ethnicity and genetic background.13,14 In addition, animal experiments have shown that taurine depletion accelerates the development of high salt–induced hypertension.15 Although taurine has been shown to lower blood pressure (BP) in several hypertensive animal models, few rigorous and long-term clinical trials have con-firmed this beneficial effect in human hypertension.9

Another key question is what is the mechanism of the antihypertensive effects of taurine supplementation?16 Recent animal and human studies have shown that taurine supple-mentation lowers BP and improves vascular function, possibly through suppression of renin–angiotensin–aldosterone system activity,17,18 augmentation of kallikrein activity in the blood and peripheral tissues,19 suppression of the renal sympathetic nervous system,9,20 diuretic and natriuretic activities, and vaso-relaxant activity.21 H

2S can regulate vascular tone through sev-

eral mechanisms, such as acting on ATP-sensitive potassium channels.22–24 A recent study has found that H

2S also affects

transient receptor potential channels (TRPCs) in mesenchy-mal stem cells and regulates calcium homeostasis.4 Our previ-ous studies have demonstrated that TRPC3-mediated calcium signaling contributes to the development of hypertension,25,26 but it is unclear whether the hypotensive effects of taurine and H

2S are associated with modulation of TRPC3 channels

in the vasculature. In this study, we investigated the effects of chronic taurine supplementation on BP and vascular func-tion in prehypertension by performing a randomized, double-blind, placebo-controlled clinical trial.

MethodsDetailed Methods are provided in the online-only Data Supplement.

Study Design and ProceduresThis study was a prospective single-center, double-blind, random-ized, placebo-controlled trial that was conducted in accordance with the CONSORT (Consolidated Standards of Reporting Trials) guidelines for the presentation of clinical trials (CONSORT 2010 Explanation and Elaboration) and the principles of the Declaration of Helsinki. The protocol was approved by the ethics committee of the Daping Hospital, Third Military Medical University. The protocol is registered in the US National Library of Medicine (http://www.ClinicalTrials.gov, identifier: NCT01816698).

Participants were recruited at the Center for Hypertension and Metabolic Diseases of Chongqing from December 2012 to December 2014. They were screened for eligibility after written informed con-sent was obtained. The prehypertension inclusion criteria for the first visit included the following: an age of between 18 and 75 years and an systolic BP (SBP) of 120 to 139 mm Hg or a diastolic BP (DBP) of 80 to 89 mm Hg, as determined by performing repeated measure-ments with a mercury sphygmomanometer. The main exclusion cri-teria included the following: clinical evidence of recent infection, pregnancy, coronary artery disease, peripheral vascular disease, cere-brovascular disease, renal dysfunction, diabetes mellitus, hyperten-sion, tumor, mental disease, the use of other medications, or being enrolled in another trial within the last 3 months.

In total, 120 untreated participants (51 men and 69 women; age, 56.75±8.26 years) and 58 age-matched normotensive control sub-jects without taurine supplementation were enrolled only as baseline

comparison in the study. These untreated participants were randomly assigned to either a placebo group or a taurine group (Figure S1 in the online-only Data Supplement). All subjects completed a standardized questionnaire administered by trained personnel on their history of cardiovascular diseases and other illnesses. All subjects were asked not to alter their usual diet over the course of the 12-week study. They all underwent standardized clinical and laboratory examinations. BP was measured by a physician using a mercury sphygmomanometer after each subject had rested for at least 5 minutes in the seated posi-tion. Three measurements were obtained at 1-minute intervals, and the average was used to define the SBP and DBP. Laboratory tests were performed after an overnight fast, including measurements of fasting plasma glucose, triglyceride, cholesterol, hepatic enzyme, uric acid, blood urea nitrogen, and serum creatine levels.

Statistical AnalysisFor all participants, we analyzed the changes from baseline (ran-domization) to 12 weeks in BP, vascular functions, biochemical and renal parameters, and other parameters. The sample size was chosen to ensure for 90% power to detect a 3-mm Hg difference in our pri-mary outcome, a change in SBP, with a 2-sided significance level of 0.05 and assuming a dropout rate of 20%, according to previous published data and a preliminary trial of prehypertensive participants. All analyses were based on intention-to-treat populations (defined as all patients who took at least 1 dose and had at least 1 efficacy measurement available after randomization), with the last value car-ried forward for missing values. Comparisons of continuous variables between the placebo and taurine groups were analyzed using the Mann–Whitney test (GraphPad Prism; La Jolla, CA). Comparisons of variables before and after treatments were analyzed using the Wilcoxon signed-rank matched pair test. The χ2 test was used for cat-egorical variables. Spearman nonparametric correlation analysis was performed to determine the relationships between BP changes and other factors. The immunoblotting results, wire myograph results, and PTI (Photon Technology International) results were compared using the Mann–Whitney test. A 2-tailed P<0.05 was considered sta-tistically significant. The data were expressed as mean±SEM or SD for normally distributed variables and median (25th and 75th percen-tiles) for non-normally distributed variables, and all the results were analyzed using SPSS 18.0.

ResultsBaseline Characteristics of ParticipantsCompared with the normal controls, the enrolled prehyperten-sive participants had higher clinic and ambulatory BPs (ABPs) and increased pulse wave velocity and postprandial blood glu-cose values. In addition, there were no significant differences in the baseline characteristics between the placebo and taurine groups (Table S1). Of 793 participants screened in the study, 120 untreated participants were randomized; of whom, 97 completed the entire study protocol and had complete data, with a loss rate of 19.2% (Figure S1). Both the taurine and pla-cebo interventions were well tolerated, and no serious adverse events were reported by any of the participants.

Chronic Taurine Supplementation Reduces BP in Prehypertensive IndividualsAdministration of taurine for 12 weeks significantly reduced BP. The clinic SBP and DBP decreased in the taurine group by 7.2 mm Hg (95% confidence interval [CI], 3.75–10.55; P<0.001) and 4.7 mm Hg (95% CI, 2.16–7.14; P<0.001), respectively, compared with the baseline values; however, these changes were not evident in the placebo group (Figure 1A and 1B; Figure S2A and S2B; Table S2). Similarly, the 24-hour ABP in the taurine group exhibited a similar pattern, with mean



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Sun et al Taurine Supplementation Improves Prehypertension 543

decreases in the SBP and DBP of 3.8 mm Hg (95% CI, 1.97–5.56; P<0.05) and 3.5 mm Hg (95% CI, 2.14–4.81; P<0.05), respectively, compared with the baseline values; however, no changes were observed in the placebo-treated group (Figure 1C and 1D; Figure S3A and S3B; Table S2; n=44 in the placebo group and n=42 in the taurine group, respectively). Further analysis revealed that taurine treatment reduced the daytime

ambulatory SBP and DBP compared with the baseline values, thereby decreasing the ambulatory SBP by 4.5 mm Hg (95% CI, 2.21–6.79 mm Hg; P<0.05) and the ambulatory DBP by 4.3 mm Hg (95% CI, 2.82–5.80 mm Hg; P<0.01; Figure S3C and S3D); however, no significant changes were observed in the placebo group. Meanwhile, the taurine treatment did not influence the nighttime ABP (Figure S3E and S3F).

Figure 1. Effect of taurine supplementation on blood pressure (BP). A and B, Clinic systolic BPs (SBPs; A) and diastolic BPs (DBPs; B) of participants treated with placebo or taurine at baseline (0 weeks, Pre) and after treatment (12 weeks, Post). The data are presented as the mean±SD; ***P<0.001, compared with baseline values. C and D, Twenty-four–hour average ambulatory BPs of the participants at 0 week and 12 weeks compared with the corresponding baseline values. n=44 in the placebo group and n=42 in the taurine group, respectively; *P<0.05. E and F, Clinic SBPs and DBPs at 0, 4, 8, and 12 weeks in the 2 groups. *P<0.05 and **P<0.01 compared with the placebo group. G and H, Comparisons of BP changes between the prehypertensive participants with high- and low-normal BPs in the taurine group. *P<0.05. ns indicates not significant.



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Chronic taurine supplementation time dependently decreased clinic BP. Compared with the placebo group, both the clinic SBP and DBP were significantly reduced at 8 and 12 weeks after taurine administration (Figure 1E and 1F; Figure S2C and S2D). Importantly, taurine supplementation for 12 weeks greatly reduced the BPs of the prehypertensive par-ticipants with high-normal BPs (SBP, 130–139/DBP, 85–89 mmHg) compared with the prehypertensive participants with low-normal BPs (SBP, 120–129 mm Hg; DBP, 80–84 mm Hg). Changes in the SBP of 10.1 mm Hg were observed in the high-normal BP group compared with changes of 3.0 mm Hg in the low-normal BP group (P<0.05; Figure 1G). However, the changes in the DBP were similar between these 2 subgroups (Figure 1H).

Taurine Supplementation Improves Vasodilation in Prehypertensive IndividualsChronic taurine supplementation significantly improved both endothelium-dependent vasodilation (flow-mediated dilation) and endothelium-independent vasodilation (nitro-glycerin-mediated dilation) by 3.2% and 4.4%, respectively, as measured via flow-mediated vasodilation using a sonog-rapher in the prehypertensive individuals. However, the ben-eficial effect of taurine supplementation on vasodilation was absent in the prehypertensive individuals treated with placebo (Figure 2A–2D).

Taurine Supplementation Elevates Plasma Levels of Taurine and H2S in Association With BP Changes in Prehypertensive IndividualsAfter treatment for 12 weeks, the plasma taurine and H

2S lev-

els were significantly higher in the prehypertensive individu-als treated with taurine (plasma H

2S level: 43.8±20.82 µmol/L

at baseline to 87.0±24.51 µmol/L after treatment; P<0.001

and plasma taurine level: 108.3±55.27 µmol/L at baseline to 142.3±62.14 µmol/L after treatment; P<0.05); however, these changes were not observed in the participants treated with placebo (Figure 3A and 3B). Furthermore, the changes in BP were negatively correlated with both the plasma H

2S and tau-

rine levels in the taurine-treated prehypertensive individuals (Figure 3C–3F), especially in the prehypertensive participants with a high-normal BP level (Figure S4A–S4C). In contrast, these associations between BP and the plasma levels of H

2S

and taurine were not observed in the participants treated with the placebo (Figure S5A–S5D).

Effects of Taurine on H2S-Synthesizing Enzymes, TRPC3, and Vascular RelaxationTo elucidate the mechanisms underlying the effects of tau-rine on BP and vascular functions, we further examined 2 key H

2S-synthesizing enzymes, CBS and CSE. We showed

that CBS and CSE were expressed in the endothelia and adventitia of mesenteric arteries (MAs) from human and aortas from mice. However, TRPC3 was mainly expressed in the media of arteries (Figure 4A and 4B). Western blotting also indicated that CBS, CSE, and TRPC3 were coexpressed in MAs from humans and aortas from Trpc3+/+ wild-type (WT) mice (Figure 4C and 4G). In addition, vascular CBS/CSE expression was upregulated in Trpc3−/− mice compared with WT mice (Figure 4C and 4D). Administration of taurine significantly upregulated CBS/CSE expression but inhibited TRPC3 expression in both aortas from spontaneously hyper-tensive rats treated with taurine and cultured human vascular tissues (Figure 4E–4J). After depletion of intracellular cal-cium storage using thapsigargin, a sarcoplasmic reticulum Ca2+-ATPase inhibitor, KCl-induced vasoconstriction was dose dependently relaxed by NaHS, a H

2S donor; however,

this effect was enhanced by a TRPC3 inhibitor, Pyr3, or by

Figure 2. Effect of taurine supplementation on vasodilation. A and B, Flow-mediated dilation (FMD) and nitroglycerin-mediated dilation (NMD). The data are presented as the mean±SD. **P<0.01 and ***P<0.001, compared with pretreatment with taurine (Pre). C and D, Changes in FMD and NMD in the 2 groups. *P<0.05 and **P<0.01, compared with the placebo group. ns indicates not significant.



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Trpc3 gene knockout (Figure 4K and 4L). These findings indicate that TRPC3 might be involved in H

2S-mediated vas-

cular relaxation.

H2S Exerts Vascular Relaxation by Targeting TRPC3-Mediated Calcium InfluxWe further examined H

2S-induced vascular relaxation, which

occurs through the targeting of TRPC3. Intact MAs were iso-lated from WT and Trpc3−/− mice. Calcium influx of intact blood vessels was measured using fluorescence techniques after depletion of intracellular calcium storage in the absence of external calcium. The phenylephrine-induced increase in calcium influx in the artery was completely abolished by the TRPC3 inhibitor Pyr3 after thapsigargin treatment (Figure S6A and S6B). Administration of NaHS significantly diminished the thapsigargin- and phenylephrine-induced increase in calcium influx in human MAs (Figure S6C–S6F). Furthermore, NaHS partially inhibited the thapsigargin-induced calcium influx in the Trpc3+/+ mice, but this effect

was absent in intact arteries isolated from the Trpc3−/− mice (Figure S6G–S6N).

DiscussionTo the best of our knowledge, this is the first randomized, double-blind, placebo-controlled clinical trial to investigate the effects of taurine supplementation in prehypertensive individuals. Furthermore, we have provided experimental evidence to facilitate elucidation of its mechanism of action. This study has revealed that oral taurine supplementation for 12 weeks significantly reduces the clinic and 24-hour ABPs in prehypertensive individuals, especially in those with high-normal BP. In addition, taurine treatment substantially pro-motes vasodilation and elevates the plasma taurine and H

2S

levels in these individuals. Furthermore, changes in BP were negatively correlated with the plasma taurine and H

2S levels.

However, these beneficial effects were absent in the prehy-pertensive individuals treated with placebo. In experimental studies, administration of taurine has been shown to enhance

Figure 3. Effects of taurine supplementation on plasma taurine and hydrogen sulfide (H2S) levels and their associations with blood pressure (BP) changes. A and B, Changes in the plasma H2S and taurine levels. The data are presented as the mean±SEM. *P<0.05 and ***P<0.001, compared with pretreatment with taurine (Pre). C–F, Correlations between BP changes and plasma H2S and taurine levels, respectively. DBP indicates diastolic BP; ns, not significant; and SBP, systolic BP.



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the expression of H2S-synthesizing enzymes (CBS/CSE) and

to reduce vascular TRPC3 expression in spontaneously hyper-tensive rats. Furthermore, the vascular relaxation induced by the H

2S donor NaHS is enhanced by TRPC3 antagonist

treatment. These findings indicate that taurine intervention improves vascular tone by targeting the H

2S-mediated inhibi-

tion of TRPC3-induced calcium influx.Taurine is a sulfur-containing amino acid that is both cheap

and nontoxic, and it is widely used as a functional dietary fac-tor. Seafood containing an abundance of taurine improves car-diovascular and metabolic diseases, such as obesity, diabetes mellitus, and hyperlipidemia. In addition, taurine has multiple

biological effects, such as protection against liver cirrhosis and antioxidative,27 anti-inflammatory, antiatherosclerotic,28,29 and antiobesity effects.30 Unfortunately, evidence that taurine supplementation reduces BP in human hypertension is incon-clusive despite the fact that multiple experimental studies have demonstrated the hypotensive effect of taurine in different hypertensive animal models.

The antihypertensive effect of taurine in humans has only been confirmed in a few clinical studies with small sample sizes (n=10–12) that were short term (7 days to 6 weeks).31 One nonrandomized placebo-controlled trial showed that oral taurine supplementation (6 g per day) for 1 week decreased the

Figure 4. Effects of taurine on cystathionine-β-synthetase (CBS)/cystathionine-γ-lyase (CSE), transient receptor potential channel 3 (TRPC3), and vascular relaxation. A and B, The immunofluorescence staining results showing that CBS/CSE (red) and TRPC3 (green) were coexpressed in mesenteric arteries (MAs) from humans and in aortas from wild-type mice. 4′,6-Diamidino-2-phenylindole (DAPI) was also stained to show the existence of nuclei (blue). C and D, The expression levels of CBS, CSE, and TRPC3 in aortas from Trpc3+/+ and Trpc3−/− mice were detected by Western blotting. The bands from 3 independent immunoblots were quantified using Image J, and the relative expression levels are shown in (D). The data are presented as the mean±SEM; *P<0.05 and ***P<0.001, compared with that of Trpc3+/+ mice. E and F, Effects of in vivo taurine supplementation on the expression levels of CBS, CSE, and TRPC3. Spontaneously hypertensive rats (SHRs) were fed 2% taurine for 12 weeks from 4-week old, and then aortas were obtained for Western blotting analysis. *P<0.05, compared with that of the normal diet (ND). G–J, Effect of in vitro taurine supplementation on the expression levels of CBS, CSE, and TRPC3. Human MAs were treated with taurine (at doses of 0, 20, and 40 mmol/L) for 24 hours. *P<0.05 and **P<0.01, compared with the vehicles. K and L, Reactivities of human and mice MAs. After contraction induced using 60 mmol/L KCl, NaHS (10–5 to 10–3 mol/L) was added to promote vasodilation, and then the percentage of vasodilation was plotted. n=4; *P<0.05. TG indicates thapsigargin.



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SBP by 9.0 mm Hg and the DBP by 4.1 mm Hg in borderline hypertensive patients.9 In addition, taurine supplementation has been shown to lower BP by ≈22 to 49 mm Hg in differ-ent experimental hypertensive rats.31 Therefore, it remains unknown whether oral taurine supplementation is beneficial for prehypertensive individuals. In this randomized, double-blind, placebo-controlled study, we showed that administra-tion of low-dose taurine (1.6 g per day) for 12 weeks can time dependently lower both clinic and ABPs and improve vascu-lar relaxation. In particular, prehypertensive individuals with high-normal BP exhibited a better response to taurine than those with low-normal BP. Our study has provided the first solid evidence of the hypotensive effect of taurine in prehy-pertensive individuals.

Epidemiological studies have shown that the plasma taurine level is lower in patients with essential hyperten-sion.12 Nara et al32 have reported that this level is decreased in spontaneously hypertensive rats in relation to the severity of hypertension. The plasma taurine level is negatively corre-lated with BP in hypertensive patients.14 Taurine deficiency in rats accelerates high salt intake–induced hypertension through renal dysfunction.15 Galloway et al33 reported that acute tau-rine treatment resulted in a 13-fold increase in the plasma taurine concentration, whereas no significant change in the muscle taurine concentration was observed. In this study, we found that chronic taurine treatment for 12 weeks resulted in an almost 1.5-fold increase of plasma taurine concentration in the prehypertensive individuals and that this increase was cor-related with a reduction in BP. In addition, the prehypertensive individuals with a high end point plasma taurine level exhib-ited a greater hypotensive response to the taurine treatment.

The manner by which taurine exerts its hypotensive effect has been studied for a long time.16 Previous studies have shown that taurine supplementation improves endothelium-dependent vasodilation through restoration of vascular redox homeostasis and improvement of nitric oxide bioavailability.34 In addition, in human studies, improvement of flow-mediated dilation has been observed in response to dietary taurine supplementation in young smokers.35 The improved vascu-lar function may facilitate the hypotensive effect and provide extra cardiovascular benefits. Available data suggest that the hypotensive effect of taurine does not occur through 1 specific mechanism but rather through multiple mechanisms.

In addition to nitric oxide and carbon monoxide, H2S,

which is another important gas transmitter, has been widely studied in the cardiovascular system in recent years,36 and its vasodilatory effects have also been reported. H

2S is endog-

enously produced from 2 sulfur-containing amino acids, l-cysteine and l-methionine, by the 2 H

2S-synthesizing

enzymes, CBS and CSE.24 Mutant mice lacking CSE display pronounced hypertension and reduced endothelium-depen-dent vasorelaxation, but this result was not confirmed by other studies.24 However, H

2S replacement has been shown to reduce

the SBP in both Cse−/− and Cse+/− mice,24 suggesting that the H

2S synthases/H

2S pathway confer protection against hyper-

tension. Taurine, as a sulfur-containing amino acid, functions in the methionine cycle and can be converted by cysteine in the presence of cysteine dioxygenase,3 whereas H

2S is syn-

thesized from the 2 sulfur-containing amino acids, l-cysteine

and l-methionine. This study has shown that taurine is prob-ably a substrate for the synthesis of H

2S to increase CBS and

CSE expression. Therefore, we assumed that a correlation may exist between taurine and H

2S. Taurine supplementation

resulted in a significant elevation in the plasma H2S level in

the prehypertensive individuals, and this elevation was cor-related with a decrease in BP in the taurine group. Using MAs from healthy human volunteers and aortas from spontaneously hypertensive rats that were fed taurine for 3 months, we fur-ther demonstrated that taurine administration upregulated the expression of vascular CBS/CSE, which caused the increased production of H

2S in blood vessels in vitro and in vivo. Thus,

other mechanisms of the vasorelaxant effect of H2S have also

been identified involving opening of the ATP-sensitive potas-sium channels,23 interaction with nitric oxide pathways, func-tioning as an endothelium-derived hyperpolarizing factor, and direct activation of protein kinase G.37 Recently, Cheang et al38 and Tian et al39 have reported that H

2S dilates blood vessels

by opening voltage-gated potassium channels in rat coronary arteries and inhibits calcium channels in rat cerebral arteries, respectively. H

2S has been demonstrated to regulate the activi-

ties of TRP channels in bone marrow mesenchymal stem cells through sulfhydration.4 Our previous work has demonstrated that TRPC3 upregulation and dysfunction in monocytes and in the vasculature from both genetically hypertensive rats and essential hypertensive patients play important roles in the pathogenesis of hypertension.25,26,40,41 In this study, we further verified that the H

2S donor NaHS inhibited phenylephrine- and

thapsigargin-induced Ca2+ influx and relaxation in MAs from human and Trpc3+/+ WT mice; however, this effect was absent in intact arteries from Trpc3−/− mice. Our work has revealed a novel unrecognized mechanism of taurine- and H

2S-induced

vasorelaxation that functions by enhancing the metabolism of sulfur-containing amino acids.

Study LimitationsThe limitation of this study is that it was not performed across multiple centers. Further studies should validate whether this beneficial effect is present in other ethnicities and popula-tions. In addition, the hypotensive effects of taurine have been reported to occur via the central nervous system,42,43 attenuation of the overactivity of the sympathetic system and increased urinary norepinephrine, and epinephrine excretion.31 An effect of taurine on BP occurring via the central nervous system cannot be excluded.31

PerspectivesPrehypertension plays an important role in the development of hypertension. Furthermore, prehypertension is closely associated with the morbidities of stroke, ischemic heart dis-ease, and renal dysfunction. Although lifestyle modifications and an angiotensin II receptor blocker have been used to treat prehypertension, poor compliance and limitations of antihy-pertensive agents are the main obstacles of treatment. This randomized, double-blind, placebo-controlled clinical trial is the first to demonstrate that taurine supplementation signifi-cantly reduces BP and improves vascular function in prehy-pertensive individuals, especially in those with high-normal BP. Furthermore, changes in BP are correlated with both the



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plasma H2S and taurine levels in the taurine-treated prehyper-

tensive individuals. We further demonstrate that the hypoten-sive effect of taurine involved the H

2S-mediated inhibition of

TRPC3-induced calcium influx. Taurine, as the most abundant, semiessential, sulfur-containing amino acid, is rich in seafood and easily consumed daily. Considering the elevated cardio-metabolic risks of large populations of prehypertensive indi-viduals, consumption of taurine-rich food may be a promising and cost-effective approach to prehypertension treatment.

AcknowledgmentsWe gratefully acknowledge the participation of all study subjects and the technical assistance of Tingbing Cao and Lijuan Wang (Chongqing Institute of Hypertension, Chongqing, China) with our experiments.

Sources of FundingThis study was funded by the National Basic Research Program of China (2012CB517806 and 2013CB531205) and the National Natural Science Foundation of China (31430042, 81370366, 91339112, 81470536, 81270392, and 91439000).

DisclosuresNone.

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Studies Collaboration. Age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies. Lancet. 2002;360:1903–1913.

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What Is New?•This randomized, double-blind, placebo-controlled clinical trial is the first

to demonstrate the hypotensive effect of taurine supplementation in pre-hypertensive individuals.

•Taurine treatment remarkably improves blood pressure and vascular function, especially in prehypertensive individuals with high-normal blood pressure.

•The hypotensive mechanism of taurine partially involves the hydrogen sulfide–mediated inhibition of transient receptor potential channel 3–in-duced calcium influx in the vasculature.

What Is Relevant?•Taurine supplementation results in a substantial, time-dependent reduc-

tion in blood pressure in prehypertensive individuals. Daily taurine sup-plementation promotes an additional decrease in blood pressure beyond that achieved with conventional lifestyle changes and pharmacotherapy.

Summary

Daily taurine supplementation is a novel strategy for lowering blood pressure in prehypertension, either as a dietary factor or in con-junction with conventional lifestyle changes.

Novelty and Significance



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ZhuZhimingXiaojing Wang, Jing Chen, Xiangru Zeng, Zhigang Zhao, Hongbo He, Daoyan Liu and

Qianqian Sun, Bin Wang, Yingsha Li, Fang Sun, Peng Li, Weijie Xia, Xunmei Zhou, Qiang Li,Prehypertension: Randomized, Double-Blind, Placebo-Controlled Study

Taurine Supplementation Lowers Blood Pressure and Improves Vascular Function in

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Online Supplement

Taurine Supplementation Lowers Blood Pressure and Improves

Vascular Function in Prehypertension

A Randomized, Double-Blind, Placebo-Controlled Study

Qianqian Sun#, Bin Wang#, Yingsha Li#, Fang Sun, Peng Li, Weijie Xia,

Xunmei Zhou, Qiang Li, Xiaojing Wang, Jing Chen, Xiangru Zeng, Zhigang

Zhao, Hongbo He, Daoyan Liu*, Zhiming Zhu*

Center for Hypertension and Metabolic Diseases, Department of Hypertension

and Endocrinology, Daping Hospital, Third Military Medical University,

Chongqing Institute of Hypertension, Chongqing 400042, China.

*Correspondence and requests for materials should be addressed to Z.Z.

([email protected]); D.L. ([email protected]).

#These authors contributed equally to this work.

Running title: Taurine supplementation improves prehypertension

Tel.: +86-23-68767849

Fax: +86-23-68705094

2

Supplemental Methods

Randomization and blinding procedures

All eligible participants were randomized at a 1:1 ratio to the taurine or placebo group. Random numbers were generated by Microsoft Excel program, and a randomized block design was used to generate six blocks with 30 patients in each block. All blind codes, including random numbers and allocation sequences, were generated, saved and sealed in opaque envelopes by an independent blind codes manager within the hospital. All patients, study team members, and analysts were masked to the treatment assignments, with the exception of the blind codes manager. The codes could be broken in the case of a serious adverse event if it was deemed essential for the treatment of the patient, but the manager did not assess the patients or participate in the writing of the report. The experimental taurine particles and matching placebo particles were identical in shape, size, and appearance to taurine and were manufactured and packaged by an independent third-party pharmacy, which is where the drugs were produced and then packaged together in cartons that were labeled with random numbers by the pharmacy according to the blind codes and correspondingly allocated as the trial drugs.

Interventions, follow-up and study outcomes

The participants received low-dose taurine (1.6 g/day) or an equivalent amount of placebo. They were told to take the study medication between 08:00 and 09:00 am. All subjects were asked not to alter their usual diet over the course of the 12-week study, and they were all scheduled for four follow-up visits at 4, 8 and 12 weeks after randomization. The primary outcomes were defined as the changes in systolic blood pressure, diastolic blood pressure and 24-hour ambulatory blood pressure. Ambulatory BP monitoring (ABPM) was recorded using Spacelabs 90217 (Spacelabs, Medical Inc., Redmond, Washington, USA). Automatic BP readings were taken every 15 minutes throughout the day (07:00-23:00) and every 30 minutes during the night (23:00-07:00). The other main outcomes were vascular functions (flow-mediated and nitroglycerin-mediated vasodilation).

Flow- and nitroglycerin-mediated vasodilation

Flow-mediated (endothelium-dependent) and nitroglycerin-mediated (endothelium-independent) vasodilation were measured by a single trained sonographer with high-resolution ultrasonography using a 7.5-MHz linear transducer on an HY 6000 system (Wuxi Haiying Electronic Medical System Co. Ltd., Jiangsu, China)1. In detail, measurements were performed on the brachial artery at 4.5 cm above the antecubital fossa before inflation of a pneumatic cuff on the upper arm to 250 mm Hg for 5 minutes and at 1 minute after cuff release. FMD was expressed as the percentage dilation from the baseline diameter to that observed at 1 minute after cuff release. Then, 0.5 mg nitroglycerin was sublingually administered, and the diameter of the brachial artery was measured. NMD was expressed as the percentage dilation from the baseline diameter to that observed after nitroglycerin administration.

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Plasma H2S measurement

Plasma hydrogen sulfide was measured by reverse-phase high-performance liquid chromatography (RP-HPLC) after derivatization with excess monobromobimane (MBB) to form a stable sulfide-dibimane derivative2. Briefly, a total of 30 μL plasma was mixed with 70 μL of 100 mmol/L Tris-HCl buffer (pH 9.5, 0.1 mmol/L DTPA), followed by the addition of 50 μL of 10 mmol/L MBB. The reaction was stopped after 30 minutes by adding 50 μL of 200 mmol/L 5-sulfosalicylic acid, and then the sample was centrifuged and the supernatant was analyzed using a Shimadzu Prominence HPLC with fluorescence detection (λex: 390 nm and λem: 475 nm) and an Eclipse XDB-C18 column.

Animal treatments

Four-week-old male spontaneously hypertensive rats (SHRs) were obtained from Charles River Laboratories (Bar Harbor, ME, USA). The rats were housed under a 12-hour day/night cycle with free access to normal food and water. The institute’s Animal Care and Use Committee approved all animal protocols. A total of six SHRs were randomly assigned to drink water with 2% taurine (w/v) or normal water starting at 8 weeks age and continuing for 12 weeks intervention period. After the intervention, the rats were sacrificed, and aortas were isolated for Western blot analysis.

Immunofluorescence staining

Human mesenteric arteries were obtained from volunteers undergoing laparotomy for other reasons after they signed informed consent forms. Human mesenteric vessels and aortas from Trpc3 wild-type mice were sectioned to 8 μm and then fixed with 10% formalin at room temperature for 60 min. The tissues were washed with PBS and then blocked with 5% bovine serum albumin for 20 min and incubated with primary antibodies (CBS from Santa Cruz Biotechnology, TRPC3 from Alomone Labs and CSE from Novus Biologicals) overnight at 4 °C. After incubation, the tissues were washed three times and incubated with fluorescent-labeled secondary antibodies (Abcam) at room temperature for 30 min. Control experiments were performed in the absence of primary antibodies. After being washed three times, the tissues were stained with DAPI staining solution at room temperature. Images were obtained with a TE2000-U Nikon eclipse microscope and analyzed with NIS-Elements imaging software (Nikon, Japan).

Western blotting

Western blotting was conducted as previously described3 using whole protein extracts prepared from human mesenteric vessels and aortas from Trpc3 wild-type mice. The primary antibodies against CBS, CSE, and TRPC3 were the same ones used for immunofluorescence staining. GAPDH was purchased from Santa Cruz Biotechnology.

Measurement of vascular reactivity

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The vascular reactivity of freshly isolated human MAs was examined using a wire myograph (Danish Myo Technology, Denmark). The vascular reactivities of human mesenteric vessels subjected to the different treatments was assessed as previously described3.

Intracellular free Ca2+ measurement

Measurement of the intracellular Ca2+ concentrations in human mesenteric arteries was performed with Fura-2AM using a PTI Fluorescence Master System (Photon Technology International, Birmingham, NJ, USA). Briefly, freshly isolated human and mouse MAs were cut into small pieces and loaded with 5 μmol/L Fura-2-AM (Invitrogen, Thermo Fisher) and 0.025% Pluronic F-127 in physiological saline solution containing 135 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 11 mM D-glucose and 10 mM HEPES, pH 7.4, for 45 minutes at 37 °C in the dark. Then, the vessel pieces were washed three times in Ca2+-free fluid as outlined above and treated with different reagents. Fluorescence was measured at 510 nm emission and at excitation wavelengths of 340 nm and 380 nm, at baseline and after treatment. Changes in the [Ca2+]i were deduced from the ratios of the transient increases in fluorescence intensity at 340 nm and 380 nm.

References

1. Zhang H, Pu Y, Chen J, Tong W, Cui Y, Sun F, Zheng Z, Li Q, Yang T, Meng C, Lu Z, Li L, Yan Z, Liu D, Zhu Z. Gastrointestinal intervention ameliorates high blood pressure through antagonizing overdrive of the sympathetic nerve in hypertensive patients and rats. J Am Heart Assoc. 2014;3:e000929.

2. Peter EA, Shen X, Shah SH, Pardue S, Glawe JD, Zhang WW, Reddy P, Akkus NI, Varma J, Kevil CG. Plasma free H2S levels are elevated in patients with cardiovascular disease. J Am Heart Assoc. 2013; 2: e0003873.

3. Sun J, Yang T, Wang P, Ma S, Zhu Z, Pu Y, Li L, Zhao Y, Xiong S, Liu D. Activation of cold-sensing transient receptor potential melastatin subtype 8 antagonizes vasoconstriction and hypertension through attenuating rhoa/rho kinase pathway. Hypertension. 2014;63:1354-

1363.

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Supplemental Tables

Table S1. Baseline characteristics of the enrolled participants

Characteristics Normal Controls

(n=58)

Placebo

(n=55)

Taurine

(n=56)

Age (Years±SD) 56.5±10.85 57.1±8.07 55.9±8.49

Male (n, %) 22, 37.93% 23, 41.82% 23, 41.07%

Clinic BP (mmHg)±SD

SBP 112.5±6.01 130.9±6.65* 130.4±6.47*

DBP 70.4±5.80 79.3±7.89* 77.8±7.63*

Ambulatory BP (mmHg)±SD

24h ASBP 107.8±7.98 123.5±11.03* 121.6±9.89*

24h ADBP 70.9±5.73 79.0±7.78* 78.0±7.96*

PWV (cm/s) (IQR)

PWV Right 1317 (1237, 1433) 1478 (1308, 1659)* 1433 (1333, 1584)†

PWV Left 1275 (1201, 1389) 1458 (1311, 1645)* 1430 (1300, 1610)*

Plasma Glucose (mmol/L)

(IQR)

Fast 5.26 (4.71, 5.75) 5.36 (4.95, 5.85) 5.28 (4.84, 5.99)

Postprandial 6.10 (5.10, 8.01) 6.87 (6.04, 8.55) 8.17 (6.86, 9.46)*

Triglycerides (mmol/L) (IQR) 0.99 (0.80, 1.40) 1.34 (0.94, 1.76)‡ 1.59 (1.14, 2.01)*

Cholesterol (mmol/L)±SD

Total 4.85±1.07 5.11±1.07 5.23±0.97

HDL-C 1.42±0.27 1.43±0.31 1.39±0.32

LDL-C 3.06±0.99 3.15±0.87 3.17±0.85

Hepatic enzymes (IU/l)

(IQR)

AST 24 (19, 28) 22 (18, 27) 23.5 (19.75, 28.5)

ALT 18 (13, 26) 17 (14, 21) 18 (14, 24)

Uric acid (μmol/L)±SD 358.5±127.0 337.2±102.2 351.0±101.4

BUN (mmol/L)±SD 4.85±1.14 5.12±1.59 5.06±1.38

Serum creatine (μmol/L)±SD 80.49±15.02 80.22±15.83 80.22±14.16

Vacular functions (IQR)

FMD (%) 10.35 (6.55, 12.48) 10.20 (7.90, 14.40) 9.50 (6.68, 13.40)

NMD (%) 13.75 (9.85, 17.45) 12.61 (10.70, 18.10) 11.65 (9.58, 14.05)

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* P<0.001, † P<0.01, ‡ P<0.05, compared with corresponding normal controls.

SD, standard deviation; IQR: Interquartile range; BP, Blood Pressure; SBP, systolic BP; DBP, diastolic BP; ASBP, ambulatory SBP; PWV, Pulse Wave Velocity; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; BUN, Blood Urea Nitrogen; FMD, Flow-mediated Dilation; NMD, Nitroglycerin-Mediated Dilation.

7

Table S2. BP levels of participants before and after treatment for 12

weeks

Characteristics Pre-Placebo Post-Placebo Pre-Taurine Post-Taurine

Clinic SBP

(mmHg) 130.8±6.30 128.2±10.08 130.0±5.64 122.8±10.57*

Clinic DBP

(mmHg) 78.7±7.51 77.4±7.10 77.7±6.87 73.1±8.66*

24-h ASBP

(mmHg) 123.3±11.08 123.0±12.06 119.4±9.37 115.6±8.35†

24-h ADBP

(mmHg) 78.3±7.38 77.7±7.30 76.0±7.97 72.5±8.22†

* P<0.001, † P<0.05, compared with corresponding pre-taurine group.

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Supplemental Figures

Figure S1

Figure S1. Screening, enrollment, randomization, and follow-up of study participants. Of the 120 randomized participants, 48 (80%) in the placebo group and 49 (81.7%) in the taurine group completed all of the follow-up visits and experimental tests. WCH, White coat hypertension.

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Figure S2

Figure S2. Effects of taurine on clinic BP. A and B, Changes in the clinic SBP and DBP in the prehypertensive individuals after treatment with placebo or taurine for 12 weeks. *P<0.05, **P<0.01. C and D, Changes in the clinic SBP and DBP throughout the intervention period in the two groups. The data are presented as the mean±SEM. *P<0.05, **P<0.01.

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Figure S3

Figure S3. Effects of taurine on 24-h ambulatory BP. A and B, Changes in the 24 h ambulatory SBP and DBP after treatment with placebo or taurine for 12 weeks. *P<0.05, **P<0.01. C to F, The daytime (7:00 to 23:00) and nighttime (23:00 to 7:00) average ambulatory BPs before (Pre) and after (Post) treatment with placebo or taurine for 12 weeks. The data are presented as the mean ± SD. *P<0.05, **P<0.01. N=44 in the placebo group and n=42 in the taurine group.

11

Figure S4

Figure S4. Correlations between the BP changes and plasma H2S and taurine levels in the prehypertensive participants with high-normal BP after treatment with taurine for 12 weeks.

12

Figure S5

Figure S5. Correlations between the BP changes and plasma H2S and taurine levels in the prehypertensive participants treated with placebo for 12 weeks.

13

Figure S6

Figure S6. Effects of H2S on vascular relaxation and its mechanisms. A, C, E, F340 nm/F380 nm fluorescence ratios of human mesenteric arteries in response to different treatments were plotted between 0 to 300 seconds (n=4). B, D, F, The summary data show the changes in the [Ca2+]i from the baseline, ***P<0.001 vs. their controls. G, I, K, and M, F340 nm/F380 nm ratios of the Trpc3 mouse mesenteric arteries in response to the different treatments were plotted between 0 to 300 seconds (n=4). H, J, L, and N, The summary data show the changes in the [Ca2+]i from the baseline. The data are presented as the mean ± SEM for four independent experiments. ***P<0.001 vs. their controls.

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Figure S7

Figure S7. A schematic illustration depicts the hypotensive mechanism of taurine supplementation and H2S regulation of vascular tone. Adminstration of taurine supplementation up-regulated H2S-synthesizing enzymes CBS / CSE expressions and producing H2S. H2S regulates the vascular tone through the opening voltage gated potassium channel, inhibits voltage dependent calcium channel and TRPC3-mediates calcium influx.

taurine supplementation lowers blood pressure and improves … · 541 p rehypertension is highly...

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